The theme of this Program Project is to understand how molecular motor work, and in particular to elucidate the structural transitions that occur in myosin during the contractile cycle. To address these elucidate the structural transitions that occur in myosin during the contractile cycle. To address these questions, the four projects bring together a wide array of state-of-the art techniques, including single molecular mechanical measurements, in vitro motility assays, fluorescence spectroscopy, protein expression in eukaryotic systems, and protein biochemistry and characterization. In Project #1, Dr. Berger will use a spectroscopic approach, both in solution and at the single molecule level, to determine how the lever arm tilts during the contractile cycle, and at the single molecule level, to determine how the lever arm tilts during the contractile cycle, and if the cleft opens and closes to mediate its affinity for actin. Dr. Warsaw (Project #2) will use the laser trap coupled with total internal reflectance microscopy to simultaneously measure the mechanics of single myosin molecules and either the nucleotide at its active site (single molecule fluorescence), or the orientation of the lever arm (fluorescence polarization). Project #3 (Dr. Trybus) will use a mutational approach coupled to biochemical and structural techniques (cryoelectron microscopy, crystallography) to decipher how the two heads of myosin cooperate in force and motion production, how the actomyosin interface changes during the powerstroke, and if ATP hydrolysis is required for priming of the lever arm. Dr. Lowey (Project #4) proposes to test the generality of domain movements at the actomyosin interface and in the lever arm, using cryoelectron microscopic techniques. It will also be determined if the orientation of actin and myosin affect the working stroke at the single molecule level. The long-term goal is to understand how each of the structural domains of myosin contributes to chemomechanical coupling, so that it becomes possible to derive a set of design principles for molecular motors.